Relapse of typhoid fever following delayed response to meropenem: A case report and review of previously published cases indicating limited clinical efficacy of meropenem for the treatment of typhoid fever

In times of emerging multi-drug resistance among Gram-negative bacteria (including Salmonella enterica, Serovar Typhi), we observed relapse of typhoid fever following delayed response to treatment with meropenem, suggestive for limited clinical efficacy of the drug. Three previously published cases supported our suspicion. Within this context, we discuss the case details with a focus on potential explanations for insufficient clinical response to meropenem (e.g. limited intracellular penetration, phenomena of tolerance and persistence). Meropenem is a last-resort antimicrobial agent for the treatment of multi-drug resistant Gram-negative infections. Reliable clinical data evaluating the efficacy of meropenem for the treatment of typhoid fever are urgently needed. Future clinical studies evaluating typhoid fever outcome should also investigate the impact of (i) intracellular penetration of antibiotics, and (ii) tolerance and persistence on outcome.

Here, we are analysing a case of relapse following treatment of typhoid fever using meropenem. The case illustrates the diagnostic and therapeutic difficulties which arise from the above-mentioned problems. Furthermore, it is the fourth case questioning the efficacy of meropenem for the treatment of typhoid fever.

Case description
A previously healthy, Caucasian, 18-year-old man presented at our Department of Emergency Medicine for watery diarrhoea, high-grade fever, and severe malaise. Stool samples performed by the family physician had been negative, including testing for Salmonella species (spp.). Five days after returning from travelling to various countries, e.g. India and Nepal, he developed fever, chills, cough, sore throat, and headaches, which lasted for 3 days before diarrhoea started. The total duration of the disease on admission was 7 days. During his travels, he experienced gastroenteritis while he was in Delhi (oral antibiotic therapy and electrolyte solution resulted in cure after 3 days), and multiple mosquito bites in malaria-endemic countries. He denied tick bites and animal contact of all kinds. Although he had received pre-travel medical advice, he did not respect alimentary precautions (he preferred vegetables and salad in local restaurants), and had refused malaria prophylaxis due to fear of side effects. The patient did not receive any vaccination against cholera or typhoid fever. Apart from signs of exsiccosis, a thorough physical examination was unremarkable. The patient was fully conscious, had a relative bradycardia (95/min), hypotension (95/60 mmHg), high-grade fever (39°C), and normal oxygen saturation. Electrocardiogram was normal and 3/9 GMS German Medical Science 2019, Vol. 17, ISSN 1612-3174 abdominal ultrasound was consistent with diagnosis of gastroenteritis. A differential blood count demonstrated a normal white blood count, aneosinophilia and discrete thrombocytopenia (126/µl, normal: 140-200/µl). Laboratory examination revealed an elevated CRP level (93 mg/dl; normal: <5 mg/dl) and slightly elevated ALT (48 U/l, normal: <40 U/l), AST (59 U/l, normal: <41 U/l) and LDH (461 U/l, normal: <250 U/l). Creatinine levels, blood gas and urine analysis were normal. Malaria was ruled out using thick smears and rapid testing. Blood, urine, and stool cultures were performed. The latter two showed no growth. Due to suspected typhoid fever, we started intravenous ceftriaxone (2 g once daily), fluid supplementation (2.500 ml per day), and oral antipyretics (750 mg metamizole four times daily). Liver enzymes increased to ALT 97 U/l and AST 83 U/l on day 8 after disease onset; to ALT 196 U/l and AST 165 U/l on day 9 after disease onset. Diarrhoea subsided to 15-20 times per day; fever subsided as well. On day 11 after disease onset, blood cultures revealed Gram-negative bacteria. By the time, the patient's condition had not improved. We suspected a Gram-negative sepsis and changed the antibiotic regime to intravenous meropenem, 1 g three times per day. One day later, Salmonella enterica Serovar Typhi was identified (susceptibility testing: Table 2), and the therapy was continued with meropenem. Liver enzymes peaked on day 12 after the onset of initial symptoms: LDH 756 U/l, ALT 544 U/l, AST 263 U/l, alkaline phosphatase (AP) 168 U/l (normal: 55-149 U/l), and Gamma-GT 196 U/l (normal: <60 U/l). Bilirubin remained normal; abdominal ultrasound displayed mild hepato-and splenomegaly. We ruled out hepatitis A/B/C/D/E (serologic tests), enterohaemorrhagic Escherichia coli, and amoebic liver disease (stool samples), and continued the treatment regime. On day 16 after disease onset (day 9 of antibiotic therapy), the patient's condition improved, both his body temperature and his liver enzymes decreased. On day 14 of treatment (4 days ceftriaxone; 10 days meropenem), the patient had fully recovered (including complete normalization of laboratory parameters). One week after treatment, 3 stool samples (obtained on 3 different days) were negative for S. Typhi. During a family visit in Dresden 14 days after completion of initial treatment, the patient was hospitalised again (Department of Infectious Diseases and Tropical Medicine, Städtisches Klinikum Dresden) for high-grade fever, crampy abdominal pain, and watery diarrhoea. Abdominal ultrasound revealed extended mesenteric lymph nodes. The colleagues ruled out schistosomiasis and HIV (serology), as well as helminthic infections, other parasites, and Clostridium difficile (stool analyses). Blood cultures revealed S. Typhi (susceptibility testing: Table 2). Urine analysis, performed because of dysuria and pollakiuria, yielded a urinary tract infection (UTI) due to Escherichia coli (4-MRGN (German Classification of Gram-negative bacteria indicating resistance to 4 clinically relevant groups of bactericidal antibiotics: cephalosporine and acylureidopenicilline antibiotics, carbapenems and fluo-roquinolones [7]), OXA-48 positive; Colony forming units: 10 6 /ml). Consequently, the colleagues administered a combination antibiotic therapy according to susceptibility testing using intravenous ceftriaxone (2 g daily dose maintained for 28 days to address relapse) and oral sulfamethoxazole/ trimethoprim (800/160 mg daily dose, maintained for 10 days to address the UTI). The patient fully recovered. Again, 3 stool samples following treatment were negative for S. Typhi. The patient has been free of relapse for 9 months.

Discussion
The case report recalls the importance of individualized pre-travel medical advice, illustrates diagnostics of fever in a returning traveller, and demonstrates that increasing multi-drug resistance among Gram-negative bacteria impairs treatment and outcome of typhoid fever. Notably, delayed response to treatment with meropenem followed by relapse challenges the efficacy of a last-resort antimicrobial agent. Overall, pre-travel medical advice of our patient was poor (no alimentary precautions, no vaccination, no malaria prophylaxis). Individualised pre-travel medical advice including vaccination against typhoid fever might have prevented the infection [27], [28]. However, protection following immunisation is limited (75%), and there is an urgent need for improved typhoid fever vaccination [4]. The patient's history as well as clinical and laboratory findings matched typhoid fever (compare: introduction section) [4]. Additionally, important differential diagnoses were ruled out by clinical and laboratory examinations [29]. Therefore, suspicion of typhoid fever and immediate administration of ceftriaxone were justified. The decision to switch antimicrobial treatment to meropenem on day 5 of treatment was based on case deterioration and the increasing prevalence of MDR and extended spectrum of ß-lactamase producing (ESBL) Gramnegative bacteria (including Salmonella spp.) in countries which our patient had travelled to (e.g. India and Nepal) [8], [9]. However, some reasons argue against this switch. First, the expected fever clearance time of typhoid fever is approximately 7 days from treatment initiation (range: 3-12 days), depending on the antibiotic used [2], [4], [30], [13], [31], [32], [33]. Second, the patient did not match sepsis criteria by the time of the regime change [34]. As meropenem is a last-resort antimicrobial agent for the treatment of multidrug-resistant Gram-negative infections [8], it would have been reasonable to wait for the results of susceptibility testing. Once the results were available (Table 2), return to ceftriaxone was indicated [3], [8]. Although reliable clinical data supporting the use of meropenem for the treatment of typhoid fever is limited to in vitro susceptibility testing and a few case reports [9], [35], [36], we completed treatment using meropenem. Indeed, the drug did not meet the expectations. A litera- Table 2: Susceptibility testing of this report and the three previously published cases indicating limited clinical efficacy of meropenem for the treatment of typhoid fever ture search revealed three other case reports which also questioned the clinical efficacy of meropenem [35], [36], [37]. The isolates of all four cases (throughout the manuscript, all four cases refer to: this report, Kleine et al. [35], Godbole et al. [36], and Lukácová et al. [37]) did not adequately respond to meropenem monotherapy, although the isolates were fully susceptible ( Table 2) [35], [36], [37]. All four isolates demonstrated ciprofloxacin resistance and two isolates were resistant to ceftriaxone as well ( Table 2). None of the patients displayed any underlying conditions (e.g. immunosuppression, adherence of bacteria to artificial material, abscesses) which might explain the inadequate response [35], [36], [37]. Godbole et al. proposed that limited intracellular penetration of meropenem may be responsible for treatment failures [36]. The observation that ciprofloxacin and azithromycin (both accumulate intracellularly, the latter even in lysosomes [38], [39]) were particularly effective against susceptible S. Typhi strains, stresses the importance of an intracellular action of the antimicrobial agent [36]. However, excellent response to treatment with meropenem was reported, too [40]. Additionally, limited intracellular penetration (more precisely, lack of intracellular accumulation) is the case for all ß-lactam-antibiotics, including amoxicillin, ampicillin, and ceftriaxone [38], [39], which have been successfully used to treat typhoid fever [2], [4], [30]. Therefore, the phenomena of tolerance and persistence (as defined by Kerster and Fortune [41]) provide alternative explanations [42]. Due to slow growth and dormancy, tolerant bacteria temporarily survive exposure to concen-trations of antimicrobial agents, which are normally lethal [42]. If only a small bacterial subpopulation demonstrates the same capability, this is termed persistence (not to be confused: an infection which is not effectively cleared in the host is also referred to as persistent) [42]. The phenomena can be inherited (e.g. tolerance mutations in a toxin-antitoxin module), or acquired (e.g. induced by antibiotics) [42], [43]. Treatment failure due to tolerance and persistence occurs, although the Minimal Inhibitory Concentration (MIC) of the antibiotic used is well below the breakpoint (matches all four cases) [42], [43]. This implicates that survival is not related to any resistance phenotype [42], [43]. Currently, there are two options to detect tolerance and persistence: determination of the minimum duration to kill 99% (MDK 99 to detect tolerance) and 99.99% (MDK 99.99 to detect persistence) of a given bacterial population [42]; another, simpler option is the Tolerance Diffusion Test (TDtest) as provided by Grefen et al. [43]. In addition, some tolerance mutations can be detected using molecular techniques (e.g. detection of a mutation in the vapBC toxin-antitoxin module) [42], [43]. Unfortunately, none of these analyses were performed for any of the four cases. However, S. Typhi meets the prerequisites of tolerance and persistence [42]: 1. phenotypic variation in host tissues, which lead to delayed eradication [44], 2. formation of antibiotic-tolerant subpopulations [45], 3. formation of nonreplicating persisters [46].
We believe that the phenomena of tolerance and persistence are most appropriate to explain the limited response to meropenem in all four cases, as well as the relapse in our case. Survival due to tolerance and/or persistence is temporary [42], [43]. Antibiotics with a short half-life time (e.g. amoxicillin, meropenem) may therefore be more likely to help bacteria evolve tolerance (which may reach 100%), compared to antibiotics with a longer half-life time (e.g. ceftriaxone, azithromycin) [42], [43]. Lukácová et al. obtained no response to several bactericidal antibiotics administered according to susceptibility testing, including meropenem [37] -perhaps because switching between bactericidal antibiotics is not suitable for overcoming tolerance and persistence [42]. Kleine et al. reported case-deterioration although they doubled the dosages of meropenem [35]. In contrast to proper resistance, which can be overcome by increasing dosages, such action does not adequately address tolerance and persistence [42], [43]. Response of the case (reported by Kleine et al. [35]) following the addition of fosfomycin on day 19 of meropenem monotherapy may be coincidental -the phenomena respond to prolonged treatment durations [42] -, or a direct effect of combination, because combination antimicrobial therapy may overcome tolerance and persistence. The efficacy of bacteriostatic antibiotics is not affected by the phenomena [42]. Accordingly, two cases responded to bacteriostatic antibiotics (chloramphenicol: Lukácová et al. [37], and azithromycin: Godbole et al. [36]) following insufficient treatment with bactericidal antibiotics, e.g. meropenem) [36], [37]. For the case reported by Godbole et al., one may also assume an effect of combination therapy (4 days meropenem alone, 10 days meropenem and azithromycin in combination) [36]. In fact, one study indicates that the combination of ceftriaxone and azithromycin reduced bacteria-and feverclearance times when compared to monotherapy [47]. Therefore, if treatment with meropenem is unavoidable, we agree with Kleine et al. and Godbole et al. that meropenem should be combined with an antimicrobial agent [35], [36] which preferably provides an intracellular mode of action [36] and a long half-life time -at least for severe typhoid fever cases [35], [36]. Our patient relapsed 14 days after completion of treatment (relapse usually occurs within up to six weeks after treatment [4]). If meropenem is as effective as ceftriaxone, our patient displayed only one risk factor for relapse (isolated ciprofloxacin resistance) out of seven risk factors described in medical literature: 1. the drug chosen for treatment (cephalosporines other than ceftriaxone > ceftriaxone > ciprofloxacin > azithromycin); 2. duration of treatment; 3. constipation on admission; 4. fever within 14 days of admission; 5. HIV co-infection; 6. infection with multi-drug resistant/ciprofloxacin resistant strains; 7. anatomical and structural abnormalities (e.g. schistosomiasis eggs, gallstones) [2], [4], [11], [12], [13], [30], [31], [32], [33].
We believe that the patient relapsed due to reactivation of dormant bacteria which disseminated from mesenteric lymph nodes, a mechanism suggested by Griffin et al. [48]. It is quite possible that meropenem does not adequately target intracellular, dormant bacteria [36], [42]. Increased treatment durations reduced relapse rates of typhoid fever patients [33]. The fact that such action is suitable for overcoming tolerance and persistence [42] supports our assumption. Furthermore: 1. on admission for relapse, mesenteric lymph nodes of our patient were markedly distended; 2. three negative stool samples indicate that the hepatobiliary system was probably not the source of relapse; 3. clinical cure and complete normalisation of laboratory parameters (including normalisation of CRP) made relapse from abscesses unlikely.
With the urinary tract infection that resulted from a multidrug resistant Escherichia coli (4-MRGN, OXA-48 positive), the case came full circle. Plasmid-encoded resistance genes are highly transmissible among Gram-negative bacteria. Since regions where multi-drug resistant Gramnegative infections frequently occur (e.g. India) largely overlap with regions where typhoid fever is endemic, we might soon be faced with the challenge of untreatable typhoid fever [8], [9].

Conclusions
The case report illustrates that emerging multi-drug resistant typhoid fever is a threat to people residing in or travelling to endemic countries. Our analysis stresses the need for reliable clinical data evaluating the efficacy of carbapenems (e.g. meropenem) for the treatment of typhoid fever, and emphasizes the importance to further investigate the impact of tolerance and persistence on treatment and outcome (e.g. correlate the results of TD tests with clinical outcome). New strategies for infection prevention (e.g. new and better vaccines) and new treatment options (e.g. new antimicrobial agents) are urgently needed.

Notes Competing interests
The authors declare that they have no competing interests.

Financial disclosure
The authors received no funding for this analysis.